Human oncoprotein Musashi-2 N-terminal RNA recognition motif backbone assignment and identification of RNA-binding pocket.

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors.

Comparative Toxicity and Metabolism of N-Acyl Homologues of Acetaminophen and Its Isomer 3'-Hydroxyacetanilide.

The hepatotoxicity of acetaminophen (APAP) is generally attributed to the formation of a reactive quinoneimine metabolite (NAPQI) that depletes glutathione and covalently binds to hepatocellular proteins. To explore the importance of the N-acyl group in APAP metabolism and toxicity, we synthesized 12 acyl side chain homologues of acetaminophen (APAP) and its 3'-regioisomer (AMAP), including the respective N-(4-pentynoyl) analogues PYPAP and PYMAP. Rat hepatocytes converted APAP, AMAP, PYPAP, and PYMAP extensively to O-glucuronide and O-sulfate conjugates in varying proportions, whereas glutathione or cysteine conjugates were observed only for APAP and PYPAP. PYPAP and PYMAP also underwent N-deacylation followed by O-sulfation and/or N-acetylation to a modest extent. The overall rates of metabolism in hepatocytes varied approximately 2-fold in the order APAP < AMAP ≈ PYPAP < PYMAP. Rat liver microsomes supplemented with NADPH and GSH converted APAP and PYPAP to their respective glutathione conjugates (formed via a reactive quinoneimine intermediate). With PYPAP only, a hydroxylated GSH conjugate was also observed. Thus, differences in biotransformation among these analogues were modest and mostly quantitative in nature. Cytotoxicity was evaluated in cultured hepatocytes by monitoring cell death using time-lapse photomicrography coupled with Hoechst 33342 and CellTox Green dyes to facilitate counting live cells vs dead cells, respectively. Progress curves for cell death and the areas under those curves showed that toxicity was markedly dependent on compound, concentration, and time. AMAP was essentially equipotent with APAP. Homologating the acyl side chain from C-2 to C-5 led to progressive increases in toxicity up to 80-fold in the para series. In conclusion, whereas N- or ring-substitution on APAP decrease metabolism and toxicity, homologating the N-acyl side chain increases metabolism about 2-fold, preserves the chemical reactivity of quinoneimine metabolites, and increases toxicity by up to 80-fold.

Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.

The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

Protein Targets of Isoniazid-Reactive Metabolites in Mouse Liver in Vivo.

Isoniazid (INH) has been a first-line drug for the treatment of tuberculosis for more than 40 years. INH is well-tolerated by most patients, but some patients develop hepatitis that can be severe in rare cases or after overdose. The mechanisms underlying the hepatotoxicity of INH are not known, but covalent binding of reactive metabolites is known to occur in animals and is suspected in human cases. A major unresolved question is the identity of the liver proteins that are modified by INH metabolites. Treating mice with INH leads to accumulation of isonicotinoyl-lysine residues on numerous proteins in the hepatic S9 fraction. Analysis of this fraction by SDS-PAGE followed by tryptic digestion of bands and LC-MS/MS revealed a single adducted peptide derived from d-dopachrome decarboxylase. When a tryptic digest of whole S9 was applied to anti-INH antibody immobilized on beads, only 12 peptides were retained, 5 of which clearly contained isonicotinoyl-lysine adducts and could be confidently assigned to 5 liver proteins. In another experiment, undigested S9 fractions from INA-treated and untreated (UT) mice were adsorbed in parallel on anti-INA beads and the retained proteins were digested and analyzed by LC-MS/MS. The INA-S9 digest showed 1 adducted peptide that was associated with a unique protein whose identity was corroborated by numerous nonadducted peptides in the digest and 13 other proteins identified only by multiple nonadducted peptides. None of these 14 proteins was associated with any peptides present in the UT-S9 fraction. Overall, we identified 7 mouse liver proteins that became adducted by INH metabolites in vivo. Of these 7 INH target proteins, only 2 have been previously reported as targets of any reactive metabolite in vivo.

Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes.

Branched amphiphilic peptide capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bilayer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4°C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Bioinformatic analysis of 302 reactive metabolite target proteins. Which ones are important for cell death?

Many low molecular weight compounds undergo biotransformation to chemically reactive metabolites (CRMs) that covalently modify cellular proteins. However, the mechanisms by which this covalent binding leads to cytotoxicity are not understood. Prior analyses of lists of target proteins sorted by functional categories or hit frequency have not proven informative. In an attempt to move beyond covalent binding, we hypothesized that xenobiotic posttranslational modification of proteins might disrupt important protein-protein interactions (PPIs) and thereby direct cells from homeostasis into cell death pathways. To test this hypothesis, we analyzed a list of 302 proteins (66% rat, 26% mouse, 5% human) known to be targeted by 41 different cytotoxic CRMs. Human orthologs of rodent proteins were found by blast sequence alignment, and their interacting partners were found using the Human Protein Reference Database. The combined set of target orthologs and partners was sorted into KEGG pathways and Gene Ontology categories. Those most highly ranked based on sorting statistics and toxicological relevance were heavily involved with intracellular signaling pathways, protein folding, unfolded protein response, and regulation of apoptosis. Detailed examination revealed that many of the categories were flagged primarily by partner proteins rather than target proteins and that a majority of these partners interacted with just a small number of proteins in the CRM target set. A similar analysis performed without the partner proteins flagged very few categories as significant. These results support the hypothesis that disruption of important PPIs may be a major mechanism contributing to CRM-induced acute cytotoxicity.

Protein targets of thioacetamide metabolites in rat hepatocytes.

Thioacetamide (TA) has long been known as a hepatotoxicant whose bioactivation requires S-oxidation to thioacetamide S-oxide (TASO) and then to the very reactive S,S-dioxide (TASO2). The latter can tautomerize to form acylating species capable of covalently modifying cellular nucleophiles including phosphatidylethanolamine (PE) lipids and protein lysine side chains. Isolated hepatocytes efficiently oxidize TA to TASO but experience little covalent binding or cytotoxicity because TA is a very potent inhibitor of the oxidation of TASO to TASO2. However, hepatocytes treated with TASO show extensive covalent binding to both lipids and proteins accompanied by extensive cytotoxicity. In this work, we treated rat hepatocytes with [(14)C]-TASO and submitted the mitochondrial, microsomal, and cytosolic fractions to 2DGE, which revealed a total of 321 radioactive protein spots. To facilitate the identification of target proteins and adducted peptides, we also treated cells with a mixture of TASO/[(13)C2D3]-TASO. Using a combination of 1DGE- and 2DGE-based proteomic approaches, we identified 187 modified peptides (174 acetylated, 50 acetimidoylated, and 37 in both forms) from a total of 88 nonredundant target proteins. Among the latter, 57 are also known targets of at least one other hepatotoxin. The formation of both amide- and amidine-type adducts to protein lysine side chains is in contrast to the exclusive formation of amidine-type adducts with PE phospholipids. Thiobenzamide (TB) undergoes the same two-step oxidative bioactivation as TA, and it also gives rise to both amide and amidine adducts on protein lysine side chains but only amidine adducts to PE lipids. Despite their similarity in functional group chemical reactivity, only 38 of 62 known TB target proteins are found among the 88 known targets of TASO. The potential roles of protein modification by TASO in triggering cytotoxicity are discussed in terms of enzyme inhibition, protein folding, and chaperone function, and the emerging role of protein acetylation in intracellular signaling and the regulation of biochemical pathways.

Metabolism and toxicity of thioacetamide and thioacetamide S-oxide in rat hepatocytes.

The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrolobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects, TA requires oxidative bioactivation, leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO(2)), which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 h. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology, and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [(14)C]-TASO is inhibited by unlabeled TA, which acts as a "cold-trap" for [(14)C]-TA and prevents its reoxidation to [(14)C]-TASO. This in turn increases the net consumption of [(14)C]-TASO despite the fact that its oxidation to TASO(2) is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA, may help explain phenomena previously interpreted as "saturation toxicokinetics" in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification.

Liver protein targets of hepatotoxic 4-bromophenol metabolites.

The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites; yet, when administered exogenously, it has negligible hepatotoxicity as compared to BB. The protein adducts of 4BP were thus labeled as nontoxic [Monks, T. J., Hinson, J. A., and Gillette, J. R. (1982) Life Sci. 30, 841-848]. To help identify which BB-derived adducts might be related to its cytotoxicity, we sought to identify the supposedly nontoxic adducts of 4BP and eliminate them from the BB target protein list. Administration of [(14)C]-4BP to phenobarbital-induced rats resulted in covalent binding of 0.25, 0.33, and 0.42 nmol equiv 4BP/mg protein in the mitochondrial, microsomal, and cytosolic fractions, respectively. These values may be compared to published values of 3-6 nmol/mg protein from a comparable dose of [(14)C]-BB. After subcellular fractionation and 2D electrophoresis, 47 radioactive spots on 2D gels of the mitochondrial, microsomal, and cytosolic fractions were excised, digested, and analyzed by LC-MS/MS. Twenty-nine of these spots contained apparently single proteins, of which 14 were nonredundant. Nine of the 14 are known BB targets. Incubating freshly isolated rat hepatocytes with 4BP (0.1-0.5 mM) produced time- and concentration-dependent increases in lactate dehydrogenase release and changes in cellular morphology. LC-MS/MS analysis of the cell culture medium revealed rapid and extensive sulfation and glucuronidation of 4BP as well as formation of a quinone-derived glutathione conjugate. Studies with 7-hydroxycoumarin, (-)-borneol, or D-(+)-galactosamine showed that inhibiting the glucuronidation/sulfation of 4BP increased the formation of a GSH-bromoquinone adduct, increased covalent binding of 4BP to hepatocyte proteins, and potentiated its cytotoxicity. Taken together, our data demonstrate that protein adduction by 4BP metabolites can be toxicologically consequential and provide a mechanistic explanation for the failure of exogenously administered 4BP to cause hepatotoxicity. Thus, the probable reason for the low toxicity of 4BP in vivo is that rapid conjugation limits its oxidation and covalent binding and thus its toxicity.

Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites.

Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO(2)). To explore possible parallels among the metabolism, covalent binding, and toxicity of TA and thiobenzamide (TB), we exposed freshly isolated rat hepatocytes to [(14)C]-TASO or [(13)C(2)D(3)]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO(2) is formed, whereas at least some of the TASO(2) can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined.

Identification of protein targets of reactive metabolites of tienilic acid in human hepatocytes.

Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [(14)C]-TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty-one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 nonredundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2 to 4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver ( Methogo, R., Dansette, P., and Klarskov, K. ( 2007 ) Int. J. Mass Spectrom. , 268 , 284 -295 ), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e., rat vs human), and still another may be the method of detection of adducted proteins (i.e., Western blot vs C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from humans, and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future.

Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide.

Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH3C(NH)SO2H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification we required both radio-labeled and stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C-14, C-13 and/or deuterium. The methods are straightforward, utilize readily available precursors and are amenable to small scale.

Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124.

Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 A cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; K(D) = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the "off-target" effect of a small molecule is mediated by an MAI mechanism.

Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners.

BACKGROUND: Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity. RESULTS: Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein). Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine) content and protein instability index (i.e., rate of turnover in vivo) were among the features most important to determining susceptibility. CONCLUSION: As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.

Ocular and systemic safety evaluation of calcium formate as a dietary supplement.

PURPOSE: The objective of this study was to perform a preliminary evaluation of the ocular and systemic safety of calcium formate, a dietary calcium supplement for prevention and management of osteoporosis. Although formate is an endogenous product of metabolism, high concentrations are associated with toxicity during methanol overdose. METHODS: In this prospective clinical trial, 12 healthy women ingested calcium formate (1,300 mg) three times a day for 14 days. Study evaluations included physical and ocular examination, extensive laboratory testing, serum calcium and formate levels, Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, color vision, visual fields, visual evoked potential (VEP), and full-field, pattern, and multifocal electroretinograms (MERG). RESULTS: The mean baseline serum level of formate was 0.572 +/- 0.06 mM. Peak serum levels and final serum formate did not differ significantly from baseline. The final concentration was 0.582 +/- 0.091 mM. Accumulation of serum formate did not occur. There was also no evidence of toxicity with calcium formate ingestion. All examinations and tests remained normal, including optic nerve and retinal function. Three subjects had mild transient symptoms attributable to any calcium formulation. CONCLUSIONS: Calcium formate is highly bioavailable and well-tolerated. Serum formate remained at basal levels and did not accumulate with repeated dosing. Systemic and ocular safety was demonstrated by objective testing. Given its high oral bioavailability, calcium formate may be a good choice for calcium supplementation in the prevention and management of osteoporosis. Further study will be needed to evaluate its long-term safety in a larger group of subjects representing more varied age, health, dietary, and nutritional status.

Filling and mining the reactive metabolite target protein database.

The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible (http://tpdb.medchem.ku.edu:8080/protein_database/) resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of "-omics" studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites.

Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo.

Thiobenzamide (TB) is a potent hepatotoxin in rats, causing dose-dependent hyperbilirubinemia, steatosis, and centrolobular necrosis. These effects arise subsequent to and appear to result from the covalent binding of the iminosulfinic acid metabolite of TB to cellular proteins and phosphatidylethanolamine lipids [ Ji et al. ( 2007) Chem. Res. Toxicol. 20, 701- 708 ]. To better understand the relationship between the protein covalent binding and the toxicity of TB, we investigated the chemistry of the adduction process and the identity of the target proteins. Cytosolic and microsomal proteins isolated from the livers of rats treated with a hepatotoxic dose of [ carboxyl- (14)C]TB contained high levels of covalently bound radioactivity (25.6 and 36.8 nmol equiv/mg protein, respectively). These proteins were fractionated by two-dimensional gel electrophoresis, and radioactive spots (154 cytosolic and 118 microsomal) were located by phosphorimaging. Corresponding spots from animals treated with a 1:1 mixture of TB and TB- d 5 were similarly separated, the spots were excised, and the proteins were digested in gel with trypsin. Peptide mass mapping identified 42 cytosolic and 24 microsomal proteins, many of which appeared in more than one spot on the gel; however, only a few spots contained more than one identifiable protein. Eighty-six peptides carrying either a benzoyl or a benzimidoyl adduct on a lysine side chain were clearly recognized by their d 0/ d 5 isotopic signature (sometimes both in the same digest). Because model studies showed that benzoyl adducts do not arise by hydrolysis of benzimidoyl adducts, it was proposed that TB undergoes S-oxidation twice to form iminosulfinic acid 4 [PhC(NH)SO 2H], which either benzimidoylates a lysine side chain or undergoes hydrolysis to 9 [PhC(O)SO 2H] and then benzoylates a lysine side chain. The proteins modified by TB metabolites serve a range of biological functions and form a set that overlaps partly with the sets of proteins known to be modified by several other metabolically activated hepatotoxins. The relationship of the adduction of these target proteins to the cytotoxicity of reactive metabolites is discussed in terms of three currently popular mechanisms of toxicity: inhibition of enzymes important to the maintenance of cellular energy and homeostasis, the unfolded protein response, and interference with kinase-based signaling pathways that affect cell survival.

X-ray snapshot of the mechanism of inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives.

The mechanism of action of a general class of mechanism-based inhibitors of serine proteases, including human neutrophil elastase (HNE), has been elucidated by determining the X-ray crystal structure of an enzyme-inhibitor complex. The captured intermediate indicates that processing of inhibitor by the enzyme generates an N-sulfonyl imine functionality that is tethered to Ser195, in accordance with the postulated mechanism of action of this class of inhibitors. The identity of the HNE-N-sulfonyl imine species was further corroborated using electrospray ionization mass spectrometry.

Covalent modification of microsomal lipids by thiobenzamide metabolites in vivo.

Thiobenzamide (TB) is hepatotoxic in rats causing centrolobular necrosis, steatosis, cholestasis, and hyperbilirubinemia. It serves as a model compound for a number of thiocarbonyl compounds that undergo oxidative bioactivation to chemically reactive metabolites. The hepatotoxicity of TB is strongly dependent on the electronic character of substituents in the meta- and para-positions, with Hammett rho values ranging from -4 to -2. On the other hand, ortho substituents that hinder nucleophilic addition to the benzylic carbon of S-oxidized TB metabolites abrogate the toxicity and protein covalent binding of TB. This strong linkage between the chemistry of TB and its metabolites and their toxicity suggests that this model is a good one for probing the overall mechanism of chemically induced biological responses. While investigating the protein covalent binding of TB metabolites, we noticed an unusually large amount of radioactivity associated with the lipid fraction of rat liver microsomes. Thin-layer chromatography showed that most of the radioactivity was contained in a single spot more polar than the neutral lipids but less polar than the phospholipid fractions. Mass spectral analyses aided by the use of synthetic standards identified the material as N-benzimidoyl derivatives of typical microsomal phosphatidylethanolamine (PE) lipids. Quantitative analysis indicated that up to 25% of total microsomal PE became modified within 5 h after a hepatotoxic dose of TB. Further studies will be required to determine the contribution of lipid modification to the hepatotoxicity of TB.